The goal of this study is to investigate tumor promoting effects of the secreted protein osteoprotegerin (OPG). OPG expression by human breast cancer cell lines in vitro inhibits TNF- related apoptosis inducing ligand (TRAIL)-mediated cell death. Breast cancer cells may produce OPG to block effects of TRAIL and resist being killed. Our preliminary data suggests OPG also promotes metastasis. The current study will investigate regulation of OPG expression in breast cancer cells, the impact of OPG on metastasis and the mechanisms whereby OPG impacts tumor cell behavior. Data generated will reveal whether OPG inhibitors should be developed as a therapy for breast cancer. Our preliminary data links OPG expression with nuclear factor kappa B (NF-?B) activity. This link will be investigated to determine if OPG mediates tumor promoting effects of NF-?B. Human breast cancer cells will be treated with NF-?B inhibitors and effects on OPG mRNA and protein expression measured. To study OPG in vivo, human breast cancer cell lines will be treated with shRNA to knockdown OPG or non-coding shRNA to generate control cells. These cells will then be used to investigate the consequences of OPG knockdown on metastasis in the chick embryo metastasis model. The chick embryo model recapitulates the stages of metastasis observed in human tumors and represents a rapid way to gain data regarding molecular factors that promote tumorigenesis. Cells will either be introduced directly onto the chick chorioallantoic membrane to allow primary tumor formation and metastasis or injected IV to specifically study metastasis. After one week incubation, metastasis of human breast cancer cells to chick tissues can be measured by real time PCR for human DNA. These studies will be performed in the presence and absence of TRAIL to reveal whether inhibition of OPG sensitizes cells to TRAIL-mediated cell death and/or if a lack of OPG reduces metastasis independent of its interaction with TRAIL. To determine the mechanism whereby OPG impacts metastasis, primary tumors from the chick model will be analyzed for changes in the expression of genes that would result in a tumor promoting effect. In addition in vitro assays will be performed to determine the impact of OPG knockdown on apoptosis, proliferation, migration and invasion in the presence and absence of TRAIL and also Receptor Activator of Nuclear Factor-kappaB ligand (RANKL), the other molecule known to bind OPG. This will allow us to determine the effect of OPG on tumor behavior in the presence of both of its characterized ligands. This study is of relevance to publi health as it will provide evidence that treatment strategies to block OPG could be used in combination with TRAIL or other types of breast cancer therapy to prevent metastasis, improve prognosis and increase survival in breast cancer patients. PUBLIC HEALTH RELEVANCE: The current application will investigate the hypothesis that the production of the protein osteoprotegerin (OPG) by breast cancer cells promotes tumor progression and metastasis. This will demonstrate whether OPG represents a molecule that could be blocked as part of targeted treatment for breast cancer. Since many patients do not respond or become resistant to current targeted therapies then identification of new therapeutic targets such as OPG will increase treatment options and improve prognosis for breast cancer patients.